Abstract (inglese)

Tetanus and botulinum neurotoxins cause neuroparalysis by inhibiting neuroexocytosis. They are composed by two main chains: the 100 kDa heavy chain (H) mediates the neurospecific binding to target cells and chaperons the entry of the 50 kDa light chain (L). After binding on the plasma membrane, these neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, as shown here for the first time by immuno-electron microscopy. The lumenal acidic pH induces a structural change of the neurotoxin molecule that becomes capable of translocating its L chain into the cytosol, via a transmembrane protein-conducting channel made by the H chain. This is the least understood step of the intoxication process primarily because it takes place inside vesicles within the cytosol. In the present study, we describe how this passage can be made accessible to investigation by making it to occur at the plasma membrane of neurons. The neurotoxin, bound to the plasma membrane of cerebellar granular neurons in the cold, was exposed to a low pH extracellular medium and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. We found that the neurotoxin has to be bound to the membrane via at least two anchorage sites in order for a productive low-pH induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT) /B, /C and /D is similar and takes place in the same slightly acidic range, which comprises that present inside synaptic vesicles. Thanks to this reliable method we have also studied the temperature dependence and the time course of TeNT, BoNT/C and BoNT/D L chain entry across the plasma membrane. The time course of translocation of the L chain varies for the three neurotoxins, but remains in the range of minutes at 37 °C, whilst it takes much longer at °20 C. BoNT/C does not enter neurons at 20 °C. Translocation also depends on the dimension of the pH gradient. These data are discussed with respect to the contribution of the membrane translocation step to the total time to paralysis and to the low toxicity of these neurotoxins in cold-blood vertebrates.
Another fundamental event along CNTs neuron intoxication process is the reduction of the interchain disulphide bond. This is a conditio sine qua non to free the catalytic part of the molecule in the cytosol of neurons. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-Thioredoxin reductase-Thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins.
BoNT/A is the most frequent cause of human botulism and at the same time is largely used in human therapy. Some evidences indicate that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been formally proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscopy, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor SV2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle transmembrane pH gradient, we show that translocation of the neurotoxin into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by one or two neurotoxin molecules.
Another important aspect regarding CNTs research is their employment in human therapy. Accordingly, BoNT/A is almost invariably used in the treatment of many human diseases characterized by hyperactivity of peripheral cholinergic nerve terminals. Unfortunately, some patients are or become resistant to it. This drawback can be overcome by using other botulinum toxins, and pre-clinical studies have been performed with different toxin serotypes. Botulinum neurotoxin type D has never been tested in human muscles in vivo. Here we show that BoNT/D is very effective upon injection in mice, on the mouse hemidiaphragm preparation and on different rat primary neuronal cultures. From these experiments, doses to be injected in human volunteers were determined. The effect of the injection into the human Extensor Digitorum Brevis muscle was assayed by measuring the compound muscle action potential at different times after injection. Botulinum toxin type D was found to be very uneffective in inducing human skeletal muscle paralysis. These results are interpreted in terms of recent reports on neuronal surface receptors of this neurotoxin and of the established double receptor model of binding.

151. de Paiva, A. et al. A role for the interchain disulfide or its participating thiols in the internalization of botulinum neurotoxin A revealed by a toxin derivative that binds to ecto-acceptors and inhibits transmitter release intracellularly. J Biol Chem. 268, 20838-20844. (1993).
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